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New technologies for amperometric biosensors.

Amperomeric-based detectors have successfully been used as personal monitors for blood glucose levels. However, there is a desire to increase the number of compounds measured in a small blood sample, the speed of detection and enhance the reliability of the measurement. Furthermore, with the increasing use of microdialysis as a clinical sampling method in metabolic medicine, paediatric medicine and neurointensive care, there is a need for rapid on-line detection of analytes such as lactate, glucose and glutamate in low microlitre volume samples. Two approaches to these problems are described. The first uses enzymes immobilized in a packed bed with electrochemical detection of a ferrocene mediator as a flow-injection assay for use with microdialysis. Results from microdialysis of the brain of freely moving rats are described. In the second approach, thin-film techniques are used to fabricate arrays of microdisk and micro line electrodes. The properties of these arrays in free solution and in a flow cell are presented together with an example using multiple arrays to identify an analyte by oxidation potential. Finally, different enzymes are entrapped onto the surface of two arrays by electrochemical polymerization of o-phenylenediamine. The resulting device detects glucose and lactate in real-time.

Animals↗

Application of flow injection--chemiluminescence to the study of radical scavenging activity in plants.

Chemiluminescence (CL) was observed during the oxidation of luminol (2 mg/L). mediated by 0.06% hydrogen peroxide (H(2)O(2)) and cytochrome c (10 mg/L). CL intensity was decreased by the presence of radical scavengers and the reduction was linearly proportional to the concentration and ability of scavengers; butylated hydroxytoluene (BHT), caffeic acid and gallic acid. The order of effectiveness as radical scavengers was gallic acid > caffeic acid > BHT, which shows that the number of hydroxyl groups (OH) in the B-ring of flavonoids plays a key role in a good radical scavenging activity. Of eight catechins obtained from green tea extracts, (-)-catechin was the least effective and (-)-epigallocatechin gallate (EGCg) showed the strongest activity. This result indicates that the stereoscopic structure between the C-3 group and the B ring of flavonoids as well as substituents at the C-3 position make a contribution to radical scavenging activity. Of the tested Chinese herbal ingredients, five species of ingredients represented more than 90% of the radical scavenging activity.

Butylated Hydroxytoluene↗

Determination of hydrogen peroxide by micro-flow injection-chemiluminescence using a coupled flow cell reactor chemiluminometer.

A novel flow cell reactor was developed for micro-flow injection determination of hydrogen peroxide (H(2)O(2)) using horseradish peroxide (HRP)-catalysed luminol chemiluminescence. The newly developed flow cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1-0.01 mL/min, resulting in increased sensitivity of detection of light. The flow cell reactor was made by packing HRP-immobilized gels into a flow cell (Teflon tube; 6 cm x 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow-through type). The HRP-immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H(2)O(2) specimens (50 microL) were injected into a stream of water delivered at a flow rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within-run reproducibility of the assay of H(2)O(2) was 2.4% (4.85 micromol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H(2)O(2) assay was influenced by the flow rates and the injection intervals of the H(2)O(2) specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3-8 mmol/L, but 0.56 mmol/L was sufficient for assay of H(2)O(2) in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H(2)O(2) (0-9.7 micromol/L) was Y = 27.5 X(2) + 394 X + 58.9 (Y = light intensity; X = concentration of H(2)O(2)) and the detection limit of H(2)O(2) was 0.2 micromol/L. This method was used to assay glucose (2.7-16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X(2) - 351 X + 1484 (Y = light intensity; X = glucose). The within-run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% (n = 5) and 5.70% (n = 9), respectively.

Enzymes, Immobilized↗

Preparation of poly(propyleneimine) dendrimer immobilized silica gel and its application as novel microcolumn packing for the on-line FI preconcentration and separation of platinum.

A G4.0 poly(propyleneimine) dendrimer immobilized silica gel (PPID-SG) was prepared with a divergent approach from gamma-aminopropyl silica gel (APSG) by repeating the reactions: 1) cyanoethylation of -NH2 and 2) reduction of -CN. Then it was successfully applied as the microcolumn packing for the on-line flow-injection (FI) preconcentration and separation and flame atomic absorption spectrometry (FAAS) determination of Pt. A limit of detection (LOD) of 0.014 microg/mL was obtained when 0.5 microg/mL Pt in chloride acid of PH = 3 was preconcentrated with a sample flow rate of 4.6 mL/min for 60s and the relative standard deviation (RSD) was 2.65%. Coexisting metal ions with concentration of 5.0 mg/mL and anion ions of 20.0 mg/ml have no interference in the determination of Pt. The proposed method was successfully applied to the determination of Pt in nickel alloy and anode slime samples.

Dendrimers↗

Separation preconcentration method for platinum and rhodium from environmental samples using a chelating resin.

A method of determining trace levels of platinum and rhodium in different samples was investigated. The method involves separation and preconcentration of the platinum and rhodium from the matrix by flow injection (FI) on-line coupled with electrothermal atomic absorption spectrometry (ETAAS) with Zeeman effect background correction. Platinum and rhodium were adsorbed on a microcolumn packed with 1,5-bis(di-2-pyridyl)methylene thiocarbohydrazide immobilized on silica gel (DPTH-gel). The sorbed metals were directly eluted with nitric acid into the graphite furnace and determined by AAS.

Animals↗

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition.

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k'1[AE] + k''1[AE][OH-]0.5, where k'1 = 0.020 +/- 0.006 s-1 and k''1 = 2.1 +/- 0.8 (L/mol)-0.5 s-1. Irreversible decomposition of the pseudobase is described by rate = k'2[AE][OH-], where k'2 = 20.1 +/- 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.

Acridines↗

Analytical applications of flow injection with chemiluminescence detection--a review.

This paper reviews the literature on analytical applications of flow injection (FI) techniques with chemiluminescence (CL) detection from 1995-1999. The focus is on the application of FI-CL to the quantitative determination of specific analytes in real sample matrices. Therefore, entries have been tabulated under the most appropriate application area, ie pharmaceutical, environmental, foods and beverages and biomedical, as defined by the matrix that has been analysed. Each table lists analytes alphabetically and gives details of the exact sample matrix, the limit of detection (as reported in the original paper) and comments on the CL reaction used.

Environmental Pollutants↗

Effect of the geometry of microfabricated flow reactors on chemiluminescent detection of epinephrine with lucigenin.

Three types of flow reactors with different lengths (12-126 mm) and widths (0.4-2.0 mm) of channel were made on the silicon chip by microfabrication techniques for the chemiluminescent (CL) detection of epinephrine (EP) with lucigenin (Luc). The volume of each CL reactor was about 10 microL. A solution containing EP and Luc and a solution containing NaOH and periodate were injected successively into each inlet of the CL reactor in the range 20-100 microL/min with a pressure-driven flow system. The intensity of light emission was dependent on the geometry of the flow reactors. These results could be explained in terms of the differences in the diffusion length of the reactants in the flow reactors. The maximum light emission were linearly correlated, with the concentrations of EP over the range from the detection limit of 5.0 x 10(-8) mol/L up to 5.0 x 10(-6) mol/L on the use of the CL reactor with the most promising geometry.

Acridines↗

Flow injection chemiluminescent assays for glycerol and triglycerides using a co-immobilized enzyme reactor.

A flow injection method for the determination of glycerol using a co-immobilized enzyme reactor containing glycerokinase and glycerol-3-phosphate oxidase is described. The hydrogen peroxide produced is monitored by using a luminol chemiluminescence reaction in the presence of catalyst such as Co(II). The detection limit (2.5 x blank noise) for glycerol is 7 x 10(-3) mmol/L with a sample throughput of 40/h. The calibration graph is linear over the range studied (0.2-1.0 mmol/L) with relative standard deviation 1.2-2.4%. The method is applied to the determination of glycerol in blood serum produced off-line from triglycerides using lipase isolated from bovine pancreas.

Animals↗

Determination of procaine hydrochloride using flow injection inhibitory chemiluminescence.

A new flow injection chemiluminescent method has been developed for the determination of procaine hydrochloride, based on the inhibition of the chemiluminescence reaction of luminol-hydrogen peroxide by procaine hydrochloride. The influence of several surfactants and beta-cyclodextrin on the chemiluminescence intensity were studied. It was found that beta-cyclodextrin enhanced the decrease in chemiluminescence intensity. The method is simple, convenient and sensitive, with a detection limit (3 sigma) of 0.08 micro g/mL. The decreased chemiluminescence intensity is linear, with the concentration of procaine hydrochloride in the range 0.2-100.0 micro g/mL and 100.0-400.0 micro g/mL. The relative standard deviation for 10 repeated measurements were 4.5% and 3.4% for 1.0 and 20.0 micro g/mL procaine hydrochloride, respectively. The method has been successfully applied to the determination of procaine hydrochloride in injection solutions of this drug.

Anesthetics, Local↗

Determination of parathion residues in rice samples using a flow injection chemiluminescence method.

A sensitive flow injection chemiluminescence method is described for the determination of parathion pesticide, based upon its direct chemiluminescence reaction with luminol and hydrogen peroxide in the presence of non-ionic surfactant polyethylene glycol 400. Under the selected experimental conditions, the concentration of parathion is proportional to the CL intensity in the range 0.02-1.0 mg/L. The detection limit was 0.008 mg/L and the relative standard deviation was 2.8% for 0.2 mg/L parathion solution (n = 11). This method was successfully applied to the determination of parathion residue in rice samples.

Flow Injection Analysis↗

A novel chemiluminescent method for determination of phloroglucinol.

It was found that the inhibition and enhancement by phloroglucinol of the chemiluminescence from the luminol-K3Fe(CN)6 system were dependent on the pH of luminol solution and the concentration of phloroglucinol. In Na2CO(3)-NaHCO3 buffer, phloroglucinol exhibited strong chemiluminescent enhancement at pH 9.4. On this basis, a flow injection method was developed for the determination of phloroglucinol. The method was simple, rapid, convenient and sensitive, with a detection limit of 2.0 x 10(-9) mol/L. It is effective for determining phloroglucinol in the range of 1.0 x 10(-5)-5.0 x 10(-9) mol/L. The relative standard deviation is 1.3% within one day and 3.2% between days for the determination of 5.0 x 10(-7) mol/L phloroglucinol. The method has been successfully used to determine phloroglucinol in environmental water, with satisfactory results.

Flow Injection Analysis↗

Determination of ampicillin and amoxycillin by flow injection chemiluminescence method based on their enhancing effects on the luminol-periodate reaction.

In this work, a new flow injection chemiluminescence method is described for the determination of ampicillin and amoxycillin. The method is based on the strong enhancing effects of these antibiotics on the luminol-periodate reaction. The present method allows the measurements of ampicillin in the range 0.02-1.0 mg/L range and amoxycillin in the range 0.1-10.0 mg/L range with the relative standard deviations within 0.8-2.0%. The sampling frequency was calculated about 90/h. The method was successfully applied to the determination of ampicillin and amoxycillin in pharmaceutical preparations. A brief discussion on the possible chemiluminescence reaction mechanism is presented.

Amoxicillin↗

Determination of sulphite using an immobilized enzyme with flow injection chemiluminescence detection.

A flow injection method is reported for the determination of sulphite-based on chemiluminescent detection. Hydrogen peroxide is produced from sulphite using on-line covalently bound immobilized sulphite oxidase packed in a mini-column, which was mixed downstream and detected via cobalt(II)-catalysed chemiluminescent oxidation of luminol. The limit of detection (2 x standard deviation of the blank) was 1 x 10(-3) mmol/L with sample throughput 60 h(-1). The calibration data was linear over the range of 0.2-1.0 mmol/L with relative standard deviation (n = 4) in the range 0.9-2.0%.

Cobalt↗

Flow injection chemiluminescent determination of tetracycline using a tris(2,2'-bipyridine)ruthenium(II)-cerium(IV) sulphate system.

A flow-injection chemiluminescence method for the determination of tetracycline was developed. The method is based on an enhancement by tetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine)ruthenium(II). In sulphuric acid medium, the chemiluminescence is generated by the continuous oxidation of tris(2,2'-bipyridine)ruthenium(II) by cerium (IV) sulphate. The light-emission intensity is greatly enhanced in the presence of tetracycline. Under the optimum conditions, the calibration curve is linear over the range 3.75 x 10(-8) g/mL-1.5 x 10(-5) g/mL for tetracycline with the linear equation: deltaINT = 205.898 x C - 20.442 (R2 = 0.9974). The detection limit is 3.27 x 10(-8) g/mL. The proposed method was also successfully used to determine tetracycline in pharmaceutical formulation (mean recovery of tetracycline, 100.7%).

2,2'-Dipyridyl↗

Flow injection chemiluminescence determination of dihydralazine sulphate based on permanganate oxidation sensitized by rhodamine B.

A novel flow injection chemiluminescence (CL) method for the determination of dihydralazine sulphate (DHZS) is described. The method is based on the CL produced during the oxidation of DHZS by acidic permanganate solution in the presence of rhodamine B. Rhodamine B is suggested as a fluorescing compound for the energy-transferred excitation. The CL emission allows quantitation of DHZS concentration in the range 5-800 ng/mL, with a detection limit of 1.9 ng/mL (3sigma). The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compares well with the high performance liquid chromatography (HPLC) method.

Acids↗

Determination of iron in blood serum using flow injection with luminol chemiluminescence detection.

A simple and rapid fl ow injection method is reported for the determination of iron in blood serum after acid digestion with HNO3 and HClO4, based on luminol CL detection in the absence of added oxidant. The detection limit (3 s) was 1.0 nmol/L with a sample throughput of 120/h. The calibration graph was linear over the range 0.001-1.0 micromol/L (r2 = 0.9974), with relative standard deviations (RSD) (n = 4) in the range 3.2-5%. The effect of interfering cations (Ca(II), Mg(II), Cu(II), Cd(II), Pb(II), Mn(II), Zn(II), Ni(II), Co(II) and Fe(III)) and anions (Cl-, SO4(2-), HCO3-, NO3-, NO2-) were studied using a luminol CL system for Fe(II) determination. The method was applied to normal blood serum and the results (1.32 +/- 0.08-1.74 +/- 0.05 mg/L) were compared with those from a spectrophotometric reference method (1.34 +/- 0.06-1.80 +/- 0.10 mg/L), which agree fairly well with the overall reference range in blood.

Borates↗

Flow injection-chemiluminescence determination of amoxycillin using potassium permanganate and formaldehyde system.

It was found that amoxycillin can react with potassium permanganate in an acidic medium to produce chemiluminescence, which is greatly enhanced by formaldehyde. The optimum conditions for this chemiluminescent reaction were studied in detail using a flow-injection system. The experimental results indicate that, under optimum conditions, the chemiluminescence intensity is linearly related to the concentration of amoxycillin in the range 5.48 x 10(-8)-2.74 x 10(-6) mol/L, with a detection limit (3sigma) of 4.1 x 10(-8) mol/L. The relative standard deviation was 1.0% at 1.1 x 10(-6) mol/L amoxycillin (n = 11 measurements). This method has the advantages of high sensitivity, fast response and ease of operation. The method was successfully applied to the determination of amoxycillin in raw medicines and capsules.

Amoxicillin↗